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J mouse
International Journalfor Parasitology. Vol.11,No. 3,pp.217-220, 1981.
Printed in Great Britain
002&7519/81/030217-04$02.00/O Pergamon Press Ltd. 0 1981 Australian Society for Parasitology
PLASMODIUM BERGHEI: EFFECT OF CARRAGEENAN THE COURSE OF INFECTION IN THE A/J MOUSE MARK
Department
A.
JAMES*
ON
and NELDA E. ALGER
of Genetics and Development, School of Life Sciences, University of Illinois, Urbana, IL 61801, U.S.A. (Received 21 October 1980)
Abstract-JAMES M. A. & ALGER N. E. 1981. Plasmodium berghei: Effect of carrageenan on the course of infection in the A/J mouse. International Journal for Parasitology 11: 217-220. Seakem-9 calcium carrageenan, a reported anti-macrophage agent, was found to confer partial immunity in mice subsequently challenged with 5 x lo5 Phzsmodium berghei NK65A parasitized erythrocytes. Transient parasitemias and significantly extended survival times were evident in carrageenantreated animals. It was suggested that carrageenan may have enhanced nonspecific cellular immunological mechanisms or affected specific immune reactions through the cytotoxicity to suppressor macrophages. INDEX KEY WORDS: Plasmodium berghei; protozoa, parasitic; malaria; infection; mouse, A/J; carrageenan; immunity.
INTRODUCTION CARRAGEENAN is a
high molecular wt sulfated polygalactose extracted from the marine alga Chondrus crispus (Smith, Cook & Neal, 1954). Carrageenan has been reported to be toxic to macrophages in vitro (Catanzaro, Schwartz & Graham, 1971; Thomson, 1978) without affecting the viability and function of lymphocytes (Lake, Bite, Schwartz & Salvaggio, 1971; Bite, Grawell, Salvaggio & Hoffman, 1972; Hanna & Leskovitz, 1973). The evidence for the cytotoxicity to macrophages in vivo remains equivocal, however (Rumjanek, Watson & Sljivic, 1977). The present research was conducted to determine the potential of carrageenan as a tool for examining malarial immunity.
obtained from The Jackson Laboratory (Bar Harbor, Maine, U.S.A.) and has been maintained by brothersister mating in the animal breeding facilities at the University of Illinois for several years. All mice were fed on Purine Rat Chow and water ad libitum. The animals were housed under controlled temperature, humidity, and light. Dose effect: injection of mice and the monitoring of infection. Thirty mice were divided into 3 experimental
Mice. Inbred A/J male mice (12 weeks of age) were used as experimental animals. The A/J strain was originally
groups, 10 mice/group. Each mouse in the first group received 3 intraperitoneal (IP) injections (2 mg carrageenam0.25 ml injection) of Seakem-9 calcium carrageenan (Marine Colloids, Inc., Springfield, New Jersey, U.S.A.). The injections were administered at 48 h intervals. In the second group, each mouse received only one 2 mg injection (IP) of carrageenan corresponding in time with the third injection of group 1. The third group of mice served as untreated control animals. Mice in all 3 groups were finally challenged IP with 5 x lo5 P. berghei NK65A uarasitized ervthrocvtes (PRBC) 24 h after the final carrageenan injection. -The ‘course of P. berghei infection was monitored by determining the parasitemia of 600 erythrocytes in daily blood films between days 3-7 post-challenge, and by recording the mortality dates of each mouse. Seakem-9 carrageenan, an unfractionated form of the original seaweed extract (Cudkowicz & Yung, 1977), was prepared for injection by suspending in saline (8 mg/ml), and then dissolved and sterilized by autoclaving at 110°C for 15 min.
*Present address and address for correspondence: Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801, U.S.A.
SpIeen wt test. Sixteen mice were randomly divided into 4 groups, 4 mice/group. The first group was given 3 IP injections of 2 mg carrageenan at 48 h intervals. Group 2 was treated identically to the first group except the second group was also challenged with 5 x lo5 P. berghei NK65A
MATERIALS
AND METHODS
The parasite. The NK65 strain of Plasmodium berghei used in this study was originally supplied by Dr. Meir Yoeli, New York University College of Medicine, New York City, U.S.A. Specifically, the NK65A deme (Alger, Branton, Harant & Silverman, 1971) of P. berghei was utilized in all experiments.
217
218
MARK
A. JAMES and NELDAE. ALGER
PRBC 24 h after the last carrageenan injection. The third group was inoculated only with live parasites (5 x lo5 NK65A PRBC). Lastly, the fourth group was utilized as a normal, untreated control group. To summarize, the mice received either (1) carrageenan only, (2) carrageenan plus parasites, (3) parasites only, or (4) remained untreated. All treatment schedules were coordinated to enable assay of all groups on the same day, that being 24 h following the final carrageenan injection for Group 1, and the sixth day post-P. berghei challenge for Groups 2 and 3. Mice in all groups were weighed and post-treatment body weights recorded. Next, body weights and excised splenic weights were determined. Mean spleen wt/body wt ratios finally were tabulated for each group. The Student’s t test was used for statistical analyses. All data are representative of 3 replicates of each experiment.
singly-treated group and untreated controls reached 18 and 22 %, respectively. Perhaps the most dramatic effect of carrageenan on P. berghei infection was that which served to extend the post-infection survival period of the mice, although all animals eventually succumbed to the infection. The cumulative per cent mortality of mice in all groups has been plotted in Fig. 2. The mean survival time (days*S.E.M.) of the respective groups was as follows: carrageenan-treated, 3 injections = 215 2, p < 0.01; carrageenan-treated, 1 injection = 12 k 1, p < 0.05 ; normal. untreated contrbls=8aO. _
_
RESULTS In the first experiment, mice were treated with either one or three 2 mg doses of carrageenan and then challenged with 5 x lo5 P. berghei NK65A PRBC. The course of P. berghei infection was monitored and compared to an infected control group (Fig. 1). Parasitemia in the treatment group which received only 1 carrageenan injection was very similar to that found in the control animals. However, the parasitemia found in the mice which received 3 injections appeared depressed and quite transient as compared to that of the other 2 groups. Peak parasitemia reached only 4% for the multiinjected group, whereas peak parasitemia for the
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FIG. 2. Cumulative percentage mortality of mice challenged IP with 5 x 10sPlasmo&n berghe; NK65A PRBC 24 h following (1) treatment with three 2 mg doses of carrageenan (injected IP at 48 h intervals), or (2) one 2 mg IP injection of carrageenan. Death also was monitored in a normal control group.
In the spleen wt test, mean spleen wt/body wt ratios were determined and recorded in Table 1. These indices of relative splenic cellularity were found to be as follows (mean values multiplied by lo3 + S.E.M.) : carrageenan plus P. berghei= 1 1 + 1, p < 0.05; P. berghei only = 9 + 1, p < 0.05 ; carrageenan only = 7 + 2; and untreated controls = 6 + 1. Days
FIG. 1. Mean daily percentage parasitemia (+ S.E.M.) of mice challenged IP with 5 x lo6 Plasmodium berghei NK65A PRBC 24 h following (1) treatment with three 2 mg doses of carrageenan (injected IP at 48 h intervals), or (2) one 2 mg IP injection of carrageenan. The third group served as infected controls.
DISCUSSION Contrary effects of reflect that a partial or parasitemia
to the oft-reported immunosuppressive carrageenan, the data presented here pretreatment with carrageenan provokes functional immunity (modified course of with a prolonged survival time) against
Immunity to P. berg/&
219
TABLE I-POST-TREATED BODY WEIGHTF,SPLEENWEIGHTS,AND SPLEENWEIGHT/BODYWEIGHTRATIX OF MICE TREATED WITH CARRAGEENAN AND/OR CHALLENGED WITH 5 x lo5 Plastn~dium berahei NK65A PRBC
Treatment
Mean body weight Mean spleen weight (9) (+ SEM) (ma) (? SEM)
Carrageenan-treated (3 1P inj., 2 mg dosage) Carrageenan-treated (3 IP inj., 2 mg dosage) and challenged with P. berghei P. berg/r&infected only Normal, untreated controls
31.4kO.5 25.9 f 1.4
203 + 53 289 f 48
26.6kO.5 30.95 1.0
240+ 18 178&-44
Mean spleen weight (g)/body weight (g) x lo3 1+2 11&l* 9+ 1* 651
*Significantly different from control values (p < 0.05). the virulent P. berghei NK65A malaria parasite. The data suggest that 3 IP injections of 2 mg Seakem-9 carrageenan best suppressed the course of infection with 5 x lo5 P. berghei NK65A PRBC (Fig. 1).
Prolongation of survival seemed to be a good criterion for determining the effect of carrageenan on the host’s response to the parasites. The effectiveness in prolonging survival of the mice was seen in the significantly greater mean survival times as compared to that of the untreated controls. Furthermore, 3 doses proved more efficacious in prolonging survival than a single treatment. It appears that Seakem-9 carrageenan, particularly when administered in three 2 mg doses, served to enhance the resistance of the A/J mouse to P. berghei NK65A. The immunological mechanisms affected by the immunopotentiating aspects of carrageenan may be either nonspecific (resistance mediated through indiscriminate phagocytosis) and/or specific (induction of immunocompetent lymphocytes) in nature. Carrageenan has been shown to increase the cellularity of the thymus-dependent area of the white pulp in the spleen (Thomson & Horne, 1976). Although carrageenan does not appear to stimulate T cell function in viva (Thomson & Home, 1975), it is known to initiate the recruitment or trapping of lymphocytes within lymphoid organs. The stimulation of splenic lymphocytes by parasite antigens could then in turn stimulate the synthesis and release of a lymphocyte-derived mononuclear cell chemotactic factor (Wyler & Gallin, 1977) which would allow for the local accumulation of phagocytic macrophages. In an attempt to determine if carrageenan tl.eatment did, in fact, promote lymphocyte and macrophage immigration into the spleen, a spleen wt test was conducted. Relative mean spleen wt/body wt ratios were calculated (Table 1); and a significant increase in the spleen weights was found in mice treated with both carrageenan and a 5 x lo5 P. berghei NK65A PRBC, as well as in mice which were treated with only P. berghei. Inasmuch as the ratios of these 2 groups are similar, experimental data show that significant cellular infiltration into the spleen was due to the P. berghei infection and not to the carrageenan treatment. It is possible that carrageenan may act as a
reticuloendothelial system (RES) stimulator. Macrophage activation through nonspecific RES stimulation has been demonstrated in rodent malaria (Martin, Einheber, Sadun & Wren, 1967; Nussenzweig, 1967; MacGregor, Sheagren & Wolff, 1969; Clark, Allison & Cox, 1976; Clark, Cox & Allison, 1977; Cottrell, Playfair & DeSousa, 1977). Bacillus Calmette-Guerin (BCG), bacterial endotoxin, nonviable Corynebacterium parvum, and Freund’s adjuvant all produce increased resistance to Plasmodium spp. in the mouse. In addition to its effects on nonspecific immunological function, carrageenan may affect macrophage immunoregulation of specific immune mechanisms. Macrophages have been shown to depress lymphocyte responses by the action of soluble suppressive factors produced by the macrophage (Nelson, 1976). Additional immunosuppressive effects of macrophages have been demonstrated as a result of the macrophage’s ability to activate suppressor T cells (Feldmann, 1974a, b; Rich & Pierce, 1974). The ablation of these immunosuppressive effects may be accomplished if carrageenan is indeed cytotoxic to macrophages in viva. Ishizaka, Otani & Morisawa (1977) found that enzyme-rich splenic macrophages from carrageenan-treated mice decreased for at least 10 days after carrageenan administration. This observation has been confirmed during preliminary investigations (James, unpublished). Carrageenan-induced cytotoxicity to suppressor macrophages may account for the enhanced immunity to P. berghci observed in the current study. Ishizaka, Otani & Morisawa (1978) reported the same type of phenomenon in characteristically low responder C57BL/6 mice. Carrageenan treatment of the mice markedly enhanced the primary and secondary responses to the hapten-carrier conjugate dinitrophenol-bovine gamma globulin (DNP-BGG). In addition, it is possible that the observed immunological phenomena are effected by the action of carrageenan on fundamental physiological processes in the host. It has been reported that carrageenan inhibits the complement system by direct inactivation of the Cl component (Boros, Rapp & Crisler, 1965; Davies, 1965), activates Hageman factor (factor XII) (Schwartz and Kellermeyer, 1969) and kinin formation (Rothschild & Gascon, 1966), and at high
MARK A. JAMESand NELDA E. ALGER
220
concentration acts as an anticoagulant (Schwartz 8~ Kellermeyer, 1969). These physiological effects may be important in preventing the formation of intracapillary thrombi and immune-complex deposits (Boonpucknavig, Boonpucknavig & Bhamarapravati, 1972), events associated with the pathogenesis of rodent malaria. Finally, carrageenan may have directly affected parasite viability and/or erythropoiesis in the host. Pre-incubation of PRBC with carrageenan does not appear to alter the parasite, however. Although the exact immunological mechanisms remain to be elucidated, carrageenan provides us a tool for future experimentation and analysis concerning the role of the macrophage in induction and expression of immune reactivity to Plasmodium. study was based on work completed for the Ph.D. thesis by the senior author at the University of Illinois, Urbana-Champaign.
Acknowledgements-This
REFERENCES ALGER N. E., BRANTONM., HARANTJ. & SILVERMAN P. H. 1971. Plasmodium berghei NK65 in the inbred A/J mouse: variations in virulence of P. berghei demes. Journal of Protozoology 18: 598-601. BICED. E., GRAWELLD. G., SALVAGGIOJ. E. &HOFFMAN E. 0. 1972. Suppression of primary immunization by carrageenan-a macrophage toxic agent. Immunological Communications 1: 615425. B~ONPUCKNAVIGS., B~~NPUCKNAVIGV. & BHAMARAPRAVATI N. 1972. Immunopathological studies of Plasmodium berghei-infected mice. Archives ofPathology 94: 323-330. BOROST., RAPP H. J. & CRISLERC. J. 1965. The interaction between carrageenan and the first component of complement. Journal of Immunology 94: 662-666. CATANZAROP. J., SCHWARTZH. J. &GRAHAM R. C. 1971. Spectrum and possible mechanism of carrageenan cytotoxicity. American Journal ofPathology64: 387-404. CLARK I. A., ALLISONA. C. & Cox F. E. G. 1976. Protection of mice against Babesia and Plasmodium with BCG. Nature, Land. 259: 309-311. CLARK I. A., Cox F. E. G. &ALLISON A. C. 1977. Protection of mice against Babesia spp. and Plasmodium spp. with killed Corynebacterium parvum. Parasitology 74: 9-18. COTTRELLB. J., PLAYFAIRJ. H. L. & DESOUSAB. 1977. Plasmodium yoelii and Plasmodium vinckei: the effects of nonspecific immunostimulation on murine malaria. Experimental Parasitology 43: 45-53. CUDKOWICZ G. & YUNG Y.P. 1977. Abrogation of resistance to foreign bone marrow grafts by carrageenans. I. Studies with the anti-macrophage agent Seakem carrageenan. Journal of Immunology 119: 483487. DAVIES G. E. 1965. Inhibition of complement by carrageenan: mode of action, effect on allergic reactions and on complement of various species. Immunology 8: 291299. FELDMANNM. 1974a. T cell suppression in vitro. I. Role in regulation of antibody responses. European Journal of Immunology 4 : 66&666.
FELDMANN M. 19746. T cell suppression in vitro. II. Nature of specific suppressive factor. European Journal of Immunology 4 : 667-674. HANNA N. & LESKOWITZS. 1973. Structural requirements for in vivo and in vitro immunogenicity in haptenspecific delayed hypersensitivity. Cellular Immunology 7: 189-197. ISHIZAKAS., OTANI S. & MORISAWA S. 1977. Effects of carrageenan on immune responses. I. Studies on the macrophage dependency of various antigens after treatment with carrageenan. Journal of Immunology 118: 1213-1218. ISHIZAKAS., OTANI S. & MORISAWA S. 1978. Effects of carrageenan on immune responses. II. A possible regulatory role of macrophages in the immune responses of low responder mice. Journal of Immunology 120: 61-65. LAKE W. W., BICE D., SCHWARTZ H. J. & SALVAGGIOJ. 1971. Suppression of in vitro antigen-induced lymphocyte transformation by carrageenan, a macrophage toxic agent. Journal of Immunology 107: 1745-1751. MACGREC~R R. R., SHEAGREN J. N. &WOLFF S. M. 1969. Endotoxin-induced modification of Plasmodium berghei infection in mice. Journal of Immunology 102: 131-139. MARTIN L. K., EINHEBER A., SADUNE. H. &WREN R. E. 1967. Effect of bacterial endotoxin on the course of Plasmodiumbergheiinfection. ExperimentalParasitology 20: 186199. NELSOND. S. (Ed.) 1976. Immunobiology of the Macrophage. Academic Press, New York. NUSSENZWEIG R. S. 1967. Increased nonspecific resistance to malaria produced by administration of killed Corynebacterium parvum. Experimental Parasitology 21: 224-231. RICH R. R. &PIERCE C. W. 1974. Biological expression of lymphocyte activation. III. Suppression of plaqueforming cell responses in vitro by supernatant fluids from concanavalin A activated cell cultures. Journal of Immunology 112: 1360-1368. ROTHSCHILDA. M. & GASCON L. A. 1966. Sulphuric esters of polysaccharides as activators of a bradykininforming system in plasma. Nature, Lond. 212: 1364. RUMJANEKV. M., WATSON S. R. & SIJIVIC V. S. 1977. A reevaluation of the role of macrophages in carrageenaninduced immunosuppression. Immunology 33: 423-432. SCHWARTZ H. J. & KELLERMEYER R. W. 1969. Carrageenan and delayed hypersensitivity. II. Activation of Hageman Factor by carrageenan and its possible significance. Proceedings of the Society of Experimental Biology and Medicine 1321 1021-1024. _ SMITH D. B.. COOK W. H. &NEAL J. L. 1954. Fractionation of carrageenan. Archives of Biochemistry and Biophysics 45: 232-233. THOMSON A. W. 1978. Carrageenan and the immune response. Biomedicine 28: 148-152. THOMSONA. W. & HORNE C. H. W. 1975. Failure of carrageenan to affect graft-versus-host reactivity in the rat. Transplantation 20: 435-437. THOMSONA. W. & HORNE C. H. W. 1976. Toxicity of various carrageenans in the mouse. British Journal of Experimental>athology 57: 455-459. WYLER D. J. & GALI~INJ. I. 1977. Snleen-derived mononuclear cell chemotactic factor in malaria infections: a possible mechanism for splenic macrophage accumulation. Journal of Immunology 118: 478-484.
Printed in Great Britain
002&7519/81/030217-04$02.00/O Pergamon Press Ltd. 0 1981 Australian Society for Parasitology
PLASMODIUM BERGHEI: EFFECT OF CARRAGEENAN THE COURSE OF INFECTION IN THE A/J MOUSE MARK
Department
A.
JAMES*
ON
and NELDA E. ALGER
of Genetics and Development, School of Life Sciences, University of Illinois, Urbana, IL 61801, U.S.A. (Received 21 October 1980)
Abstract-JAMES M. A. & ALGER N. E. 1981. Plasmodium berghei: Effect of carrageenan on the course of infection in the A/J mouse. International Journal for Parasitology 11: 217-220. Seakem-9 calcium carrageenan, a reported anti-macrophage agent, was found to confer partial immunity in mice subsequently challenged with 5 x lo5 Phzsmodium berghei NK65A parasitized erythrocytes. Transient parasitemias and significantly extended survival times were evident in carrageenantreated animals. It was suggested that carrageenan may have enhanced nonspecific cellular immunological mechanisms or affected specific immune reactions through the cytotoxicity to suppressor macrophages. INDEX KEY WORDS: Plasmodium berghei; protozoa, parasitic; malaria; infection; mouse, A/J; carrageenan; immunity.
INTRODUCTION CARRAGEENAN is a
high molecular wt sulfated polygalactose extracted from the marine alga Chondrus crispus (Smith, Cook & Neal, 1954). Carrageenan has been reported to be toxic to macrophages in vitro (Catanzaro, Schwartz & Graham, 1971; Thomson, 1978) without affecting the viability and function of lymphocytes (Lake, Bite, Schwartz & Salvaggio, 1971; Bite, Grawell, Salvaggio & Hoffman, 1972; Hanna & Leskovitz, 1973). The evidence for the cytotoxicity to macrophages in vivo remains equivocal, however (Rumjanek, Watson & Sljivic, 1977). The present research was conducted to determine the potential of carrageenan as a tool for examining malarial immunity.
obtained from The Jackson Laboratory (Bar Harbor, Maine, U.S.A.) and has been maintained by brothersister mating in the animal breeding facilities at the University of Illinois for several years. All mice were fed on Purine Rat Chow and water ad libitum. The animals were housed under controlled temperature, humidity, and light. Dose effect: injection of mice and the monitoring of infection. Thirty mice were divided into 3 experimental
Mice. Inbred A/J male mice (12 weeks of age) were used as experimental animals. The A/J strain was originally
groups, 10 mice/group. Each mouse in the first group received 3 intraperitoneal (IP) injections (2 mg carrageenam0.25 ml injection) of Seakem-9 calcium carrageenan (Marine Colloids, Inc., Springfield, New Jersey, U.S.A.). The injections were administered at 48 h intervals. In the second group, each mouse received only one 2 mg injection (IP) of carrageenan corresponding in time with the third injection of group 1. The third group of mice served as untreated control animals. Mice in all 3 groups were finally challenged IP with 5 x lo5 P. berghei NK65A uarasitized ervthrocvtes (PRBC) 24 h after the final carrageenan injection. -The ‘course of P. berghei infection was monitored by determining the parasitemia of 600 erythrocytes in daily blood films between days 3-7 post-challenge, and by recording the mortality dates of each mouse. Seakem-9 carrageenan, an unfractionated form of the original seaweed extract (Cudkowicz & Yung, 1977), was prepared for injection by suspending in saline (8 mg/ml), and then dissolved and sterilized by autoclaving at 110°C for 15 min.
*Present address and address for correspondence: Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801, U.S.A.
SpIeen wt test. Sixteen mice were randomly divided into 4 groups, 4 mice/group. The first group was given 3 IP injections of 2 mg carrageenan at 48 h intervals. Group 2 was treated identically to the first group except the second group was also challenged with 5 x lo5 P. berghei NK65A
MATERIALS
AND METHODS
The parasite. The NK65 strain of Plasmodium berghei used in this study was originally supplied by Dr. Meir Yoeli, New York University College of Medicine, New York City, U.S.A. Specifically, the NK65A deme (Alger, Branton, Harant & Silverman, 1971) of P. berghei was utilized in all experiments.
217
218
MARK
A. JAMES and NELDAE. ALGER
PRBC 24 h after the last carrageenan injection. The third group was inoculated only with live parasites (5 x lo5 NK65A PRBC). Lastly, the fourth group was utilized as a normal, untreated control group. To summarize, the mice received either (1) carrageenan only, (2) carrageenan plus parasites, (3) parasites only, or (4) remained untreated. All treatment schedules were coordinated to enable assay of all groups on the same day, that being 24 h following the final carrageenan injection for Group 1, and the sixth day post-P. berghei challenge for Groups 2 and 3. Mice in all groups were weighed and post-treatment body weights recorded. Next, body weights and excised splenic weights were determined. Mean spleen wt/body wt ratios finally were tabulated for each group. The Student’s t test was used for statistical analyses. All data are representative of 3 replicates of each experiment.
singly-treated group and untreated controls reached 18 and 22 %, respectively. Perhaps the most dramatic effect of carrageenan on P. berghei infection was that which served to extend the post-infection survival period of the mice, although all animals eventually succumbed to the infection. The cumulative per cent mortality of mice in all groups has been plotted in Fig. 2. The mean survival time (days*S.E.M.) of the respective groups was as follows: carrageenan-treated, 3 injections = 215 2, p < 0.01; carrageenan-treated, 1 injection = 12 k 1, p < 0.05 ; normal. untreated contrbls=8aO. _
_
RESULTS In the first experiment, mice were treated with either one or three 2 mg doses of carrageenan and then challenged with 5 x lo5 P. berghei NK65A PRBC. The course of P. berghei infection was monitored and compared to an infected control group (Fig. 1). Parasitemia in the treatment group which received only 1 carrageenan injection was very similar to that found in the control animals. However, the parasitemia found in the mice which received 3 injections appeared depressed and quite transient as compared to that of the other 2 groups. Peak parasitemia reached only 4% for the multiinjected group, whereas peak parasitemia for the
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FIG. 2. Cumulative percentage mortality of mice challenged IP with 5 x 10sPlasmo&n berghe; NK65A PRBC 24 h following (1) treatment with three 2 mg doses of carrageenan (injected IP at 48 h intervals), or (2) one 2 mg IP injection of carrageenan. Death also was monitored in a normal control group.
In the spleen wt test, mean spleen wt/body wt ratios were determined and recorded in Table 1. These indices of relative splenic cellularity were found to be as follows (mean values multiplied by lo3 + S.E.M.) : carrageenan plus P. berghei= 1 1 + 1, p < 0.05; P. berghei only = 9 + 1, p < 0.05 ; carrageenan only = 7 + 2; and untreated controls = 6 + 1. Days
FIG. 1. Mean daily percentage parasitemia (+ S.E.M.) of mice challenged IP with 5 x lo6 Plasmodium berghei NK65A PRBC 24 h following (1) treatment with three 2 mg doses of carrageenan (injected IP at 48 h intervals), or (2) one 2 mg IP injection of carrageenan. The third group served as infected controls.
DISCUSSION Contrary effects of reflect that a partial or parasitemia
to the oft-reported immunosuppressive carrageenan, the data presented here pretreatment with carrageenan provokes functional immunity (modified course of with a prolonged survival time) against
Immunity to P. berg/&
219
TABLE I-POST-TREATED BODY WEIGHTF,SPLEENWEIGHTS,AND SPLEENWEIGHT/BODYWEIGHTRATIX OF MICE TREATED WITH CARRAGEENAN AND/OR CHALLENGED WITH 5 x lo5 Plastn~dium berahei NK65A PRBC
Treatment
Mean body weight Mean spleen weight (9) (+ SEM) (ma) (? SEM)
Carrageenan-treated (3 1P inj., 2 mg dosage) Carrageenan-treated (3 IP inj., 2 mg dosage) and challenged with P. berghei P. berg/r&infected only Normal, untreated controls
31.4kO.5 25.9 f 1.4
203 + 53 289 f 48
26.6kO.5 30.95 1.0
240+ 18 178&-44
Mean spleen weight (g)/body weight (g) x lo3 1+2 11&l* 9+ 1* 651
*Significantly different from control values (p < 0.05). the virulent P. berghei NK65A malaria parasite. The data suggest that 3 IP injections of 2 mg Seakem-9 carrageenan best suppressed the course of infection with 5 x lo5 P. berghei NK65A PRBC (Fig. 1).
Prolongation of survival seemed to be a good criterion for determining the effect of carrageenan on the host’s response to the parasites. The effectiveness in prolonging survival of the mice was seen in the significantly greater mean survival times as compared to that of the untreated controls. Furthermore, 3 doses proved more efficacious in prolonging survival than a single treatment. It appears that Seakem-9 carrageenan, particularly when administered in three 2 mg doses, served to enhance the resistance of the A/J mouse to P. berghei NK65A. The immunological mechanisms affected by the immunopotentiating aspects of carrageenan may be either nonspecific (resistance mediated through indiscriminate phagocytosis) and/or specific (induction of immunocompetent lymphocytes) in nature. Carrageenan has been shown to increase the cellularity of the thymus-dependent area of the white pulp in the spleen (Thomson & Horne, 1976). Although carrageenan does not appear to stimulate T cell function in viva (Thomson & Home, 1975), it is known to initiate the recruitment or trapping of lymphocytes within lymphoid organs. The stimulation of splenic lymphocytes by parasite antigens could then in turn stimulate the synthesis and release of a lymphocyte-derived mononuclear cell chemotactic factor (Wyler & Gallin, 1977) which would allow for the local accumulation of phagocytic macrophages. In an attempt to determine if carrageenan tl.eatment did, in fact, promote lymphocyte and macrophage immigration into the spleen, a spleen wt test was conducted. Relative mean spleen wt/body wt ratios were calculated (Table 1); and a significant increase in the spleen weights was found in mice treated with both carrageenan and a 5 x lo5 P. berghei NK65A PRBC, as well as in mice which were treated with only P. berghei. Inasmuch as the ratios of these 2 groups are similar, experimental data show that significant cellular infiltration into the spleen was due to the P. berghei infection and not to the carrageenan treatment. It is possible that carrageenan may act as a
reticuloendothelial system (RES) stimulator. Macrophage activation through nonspecific RES stimulation has been demonstrated in rodent malaria (Martin, Einheber, Sadun & Wren, 1967; Nussenzweig, 1967; MacGregor, Sheagren & Wolff, 1969; Clark, Allison & Cox, 1976; Clark, Cox & Allison, 1977; Cottrell, Playfair & DeSousa, 1977). Bacillus Calmette-Guerin (BCG), bacterial endotoxin, nonviable Corynebacterium parvum, and Freund’s adjuvant all produce increased resistance to Plasmodium spp. in the mouse. In addition to its effects on nonspecific immunological function, carrageenan may affect macrophage immunoregulation of specific immune mechanisms. Macrophages have been shown to depress lymphocyte responses by the action of soluble suppressive factors produced by the macrophage (Nelson, 1976). Additional immunosuppressive effects of macrophages have been demonstrated as a result of the macrophage’s ability to activate suppressor T cells (Feldmann, 1974a, b; Rich & Pierce, 1974). The ablation of these immunosuppressive effects may be accomplished if carrageenan is indeed cytotoxic to macrophages in viva. Ishizaka, Otani & Morisawa (1977) found that enzyme-rich splenic macrophages from carrageenan-treated mice decreased for at least 10 days after carrageenan administration. This observation has been confirmed during preliminary investigations (James, unpublished). Carrageenan-induced cytotoxicity to suppressor macrophages may account for the enhanced immunity to P. berghci observed in the current study. Ishizaka, Otani & Morisawa (1978) reported the same type of phenomenon in characteristically low responder C57BL/6 mice. Carrageenan treatment of the mice markedly enhanced the primary and secondary responses to the hapten-carrier conjugate dinitrophenol-bovine gamma globulin (DNP-BGG). In addition, it is possible that the observed immunological phenomena are effected by the action of carrageenan on fundamental physiological processes in the host. It has been reported that carrageenan inhibits the complement system by direct inactivation of the Cl component (Boros, Rapp & Crisler, 1965; Davies, 1965), activates Hageman factor (factor XII) (Schwartz and Kellermeyer, 1969) and kinin formation (Rothschild & Gascon, 1966), and at high
MARK A. JAMESand NELDA E. ALGER
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concentration acts as an anticoagulant (Schwartz 8~ Kellermeyer, 1969). These physiological effects may be important in preventing the formation of intracapillary thrombi and immune-complex deposits (Boonpucknavig, Boonpucknavig & Bhamarapravati, 1972), events associated with the pathogenesis of rodent malaria. Finally, carrageenan may have directly affected parasite viability and/or erythropoiesis in the host. Pre-incubation of PRBC with carrageenan does not appear to alter the parasite, however. Although the exact immunological mechanisms remain to be elucidated, carrageenan provides us a tool for future experimentation and analysis concerning the role of the macrophage in induction and expression of immune reactivity to Plasmodium. study was based on work completed for the Ph.D. thesis by the senior author at the University of Illinois, Urbana-Champaign.
Acknowledgements-This
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